The phosphorylation of proteins by protein kinases plays an important role in the signal transduction between cells and the control of cell proliferation and differentiation (J. M. Bishop, Cell, 64, 235-248 (1991)). This reaction is reversibly controlled by the protein phosphatase-catalyzed dephosphorylation reaction. Accordingly, it is important to study protein phosphatases as well as to study protein kinases.
For the study of protein phosphatases, substrates and methods for measuring its activity are necessary. Substrates and measuring methods which have hitherto been used are as follows:
1) Using a phosphorylated amino acid-containing peptide derivative as a substrate, the release of phosphate by the action of a protein phosphatase is determined by colorimetry (H. Cho et al., Biochemistry, 30, 6210-6216 (1991)). The detection limit of phosphate by this method is about 1 nanomole. This method has a drawback that endogenous phosphate in the enzyme samples hinders the assay.
2) Using a phosphorylated amino acid-containing peptide derivative as a substrate, the absorbance or the fluorescence intensity increasing with the dephosphorylation by the action of a protein phosphatase is measured (Z. Zhao et al., Analytical Biochemistry, 201, 361-366 (1992)). The detection limit of phosphate according to this method is about 1 nanomole for the absorbance method, and about 10 picomoles for the fluorescence method. In the measurement of living samples, accurate measurement is hardly possible because of many kinds of ingredients contained therein.
3) Using a peptide derivative or a protein labelled with radioactive phosphoric acid as a substrate, the radioactivity of phosphate released by the action of a protein phosphatase is measured (N. K. Tonks et al., J. Biological Chemistry, 263, 6722-6730 (1988)). Using radioactive phosphoric acid having the specific activity of 1,000 counts per minute per picomole, the detection limit of phosphate according to this method is about 0.1 picomole. This method has the advantage that slight enzyme activity can be measured because of its high detection sensitivity. However, it has the disadvantage that the preparation of the substrate is complicated and the substrate can not be stored for a long period of time because of the decay of the radioactivity.
As described above, each of the methods for measuring protein phosphatase activity which have hitherto been employed has its merits and demerits, and hence is not necessarily satisfactory. Therefore, the development of measuring methods which are easy, high in accuracy and safe without use of radioactive substances has been desired.